Modeling the reactive cholangiocyte phenotype in cholangiopathies using human biliary organoids for evaluation of novel therapeutic compounds

Introduction Cholangiocytes actively participate in biliary inflammation during cholestatic disorders such as primary sclerosing cholangitis (PSC) by acquiring the so called “reactive cholangiocyte phenotype”. Multifactorial pathways involving bacterial metabolites, accumulating bile acids (BAs) or proinflammatory cytokines play significant roles in cholangiocyte activation. This study aims to (i) characterize the molecular pathways underlying cholangiocyte reactivity using human organoid cultures, (ii) explore the therapeutic effects of the novel hydrophilic, therapeutic BAs norucholic acid (norUDCA/NCA) and tetrahydroxylated BAs (THBA) on activated cholangiocytes.

Material and Methods Extrahepatic human cholangiocyte organoids (ECOs) were challenged either with a mixture of 10ng/ml Lipopolysaccharide (LPS) and 100U/L Interferon-γ (IFNγ) or 50ng/ml IL-17A and 2ng/ml TNFa for various time periods (1 – 24h). To evaluate the therapeutic potential of hydrophilic BAs, ECOs were treated with 250μM norUDCA/NCA or 0,1μM THBA alone, or in combination to the inflammatory trigger. Gene expression profiles of inflammatory mediators (Ccl2, Cxcl2, Cxcl10, Ccl20, IL-8) and transcription factors (EGR-1, NFκB1/2, REL, RORγT) were analyzed by qPCR.

Results mRNA-expression profiling demonstrated that the combination of IL-17A and TNFα exerted synergistic effects, elevating Ccl20 128-fold, significantly above single cytokine (7-fold IL-17a; 21,1-fold TNFa) treatments (P<0,0001). Co-treatment with 0.1 μM THBA significantly attenuated the IL-17A/TNFα-mediated inflammatory response, reducing Ccl2 (by 23%, P=0,013), Cxcl2 (60%, P=0,015) Il-8 (53%, P=0,039) and Ccl20 (40%, P=0,027) expression. NCA also demonstrated anti-inflammatory effects, decreasing Ccl2 (22%, P=0,015), and Cxcl2 (45%, P=0,05). LPS/IFNγ stimulation induced pro-inflammatory chemokine expression in ECOs (5-fold Cxcl2 (P=0,005), 12,3-fold Ccl2 (P=0,003), and 764,9-fold Cxcl10 (P=0,0008)). Co-treatment with 0.1 μM THBA demonstrated anti-inflammatory effects by reducing Ccl2 (P=0,0019) and Cxcl2 (P=0,0198) back to near-baseline levels and Cxcl10 by about 50% (P=0,001).

Conclusion The BAs THBA & NCA can ameliorate the reactive cholangiocyte phenotype induced by LPS/IFNγ and IL-17A/TNFα, arguing for potentially therapeutic effects in cholestatic liver diseases such as PSC.

Publikationsverlauf

Artikel online veröffentlicht:
13. Mai 2025